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Gene Function Rather than Reproductive Mode Drives the Evolution of RNA Helicases in Sexual and Apomictic Boechera.

ABSTRACT: In higher plants, sexual and asexual reproductions through seeds (apomixis) have evolved as alternative strategies. Evolutionary advantages leading to coexistence of both reproductive modes are currently not well understood. It is expected that accumulation of deleterious mutations leads to a rapid elimination of apomictic lineages from populations. In this line, apomixis originated repeatedly, likely from deregulation of the sexual pathway, leading to alterations in the development of reproductive lineages (germlines) in apomicts as compared with sexual plants. This potentially involves mutations in genes controlling reproduction. Increasing evidence suggests that RNA helicases are crucial regulators of germline development. To gain insights into the evolution of 58 members of this diverse gene family in sexual and apomictic plants, we applied target enrichment combined with next-generation sequencing to identify allelic variants from 24 accessions of the genus Boechera, comprising sexual, facultative, and obligate apomicts. Interestingly, allelic variants from apomicts did not show consistently increased mutation frequency. Either sequences were highly conserved in any accession, or allelic variants preferentially harbored mutations in evolutionary less conserved C- and N-terminal domains, or presented high mutation load independent of the reproductive mode. Only for a few genes allelic variants harboring deleterious mutations were only identified in apomicts. To test if high sequence conservation correlates with roles in fundamental cellular or developmental processes, we analyzed Arabidopsis thaliana mutant lines in VASA-LIKE (VASL), and identified pleiotropic defects during ovule and reproductive development. This indicates that also in apomicts mechanisms of selection are in place based on gene function.

SUBMITTER: Kiefer M

PROVIDER: S-EPMC7250504 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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Gene Function Rather than Reproductive Mode Drives the Evolution of RNA Helicases in Sexual and Apomictic Boechera.

Kiefer Markus M Nauerth Berit H BH Volkert Christopher C Ibberson David D Loreth Anna A Schmidt Anja A

Genome biology and evolution 20200501 5

In higher plants, sexual and asexual reproductions through seeds (apomixis) have evolved as alternative strategies. Evolutionary advantages leading to coexistence of both reproductive modes are currently not well understood. It is expected that accumulation of deleterious mutations leads to a rapid elimination of apomictic lineages from populations. In this line, apomixis originated repeatedly, likely from deregulation of the sexual pathway, leading to alterations in the development of reproduct ...[more]

PMID: 32302391

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Project description:BackgroundApomixis, a natural form of asexual seed production in plants, is considered to have great biotechnological potential for agriculture. It has been hypothesised that de-regulation of the sexual developmental pathway could trigger apomictic reproduction. The genus Boechera represents an interesting model system for understanding apomixis, having both sexual and apomictic genotypes at the diploid level. Quantitative qRT-PCR is the most extensively used method for validating genome-wide gene expression analyses, but in order to obtain reliable results, suitable reference genes are necessary. In this work we have evaluated six potential reference genes isolated from a 454 (FLX) derived cDNA library of Boechera. RNA from live microdissected ovules and anthers at different developmental stages, as well as vegetative tissues of apomictic and sexual Boechera, were used to validate the candidates.ResultsBased on homologies with Arabidopsis, six genes were selected from a 454 cDNA library of Boechera: RPS18 (Ribosomal sub protein 18), Efalpha1 (Elongation factor 1 alpha), ACT 2 (Actin2), UBQ (polyubiquitin), PEX4 (Peroxisomal ubiquitin conjugating enzyme) and At1g09770.1 (Arabidopsis thaliana cell division cycle 5). Total RNA was extracted from 17 different tissues, qRT-PCRs were performed, and raw Ct values were analyzed for primer efficiencies and gene ratios. The geNorm and normFinder applications were used for selecting the most stable genes among all tissues and specific tissue groups (ovule, anthers and vegetative tissues) in both apomictic and sexual plants separately. Our results show that BoechRPS18, BoechEfα1, BoechACT2 and BoechUBQ were the most stable genes. Based on geNorm, the combinations of BoechRPS18 and BoechEfα1 or BoechUBQ and BoechEfα1 were the most stable in the apomictic plant, while BoechRPS18 and BoechACT2 or BoechUBQ and BoechACT2 performed best in the sexual plant. When subgroups of tissue samples were analyzed, different optimal combinations were identified in sexual ovules (BoechUBQ and BoechEfα1), in anthers from both reproductive systems (BoechACT2 and BoechEfα1), in apomictic vegetative tissues (BoechEfα1 and BoechACT2) and sexual vegetative tissues (BoechRPS18 and BoechEfα1). NormFinder ranked BoechACT2 as the most stable in the apomictic plant, while BoechRPS18 was the best in the sexual plant. The subgroups analysis identified the best gene for both apomictic and sexual ovules (BoechRPS18), for anthers from both reproductive system (BoechEfα1) and for apomictic and vegetative tissues (BoechACT2 and BoechRPS18 respectively)ConclusionsFrom a total of six tested genes, BoechRPS18, BoechEfα1, BoechACT2 and BoechUBQ showed the best stability values. We furthermore provide detailed information for the accurate normalization of specific tissue gene expression analyses of apomictic and sexual Boechera.

Project description:BackgroundApomixis or asexual seed formation represents a potentially important agronomic trait whose introduction into crop plants could be an effective way to fix and perpetuate a desirable genotype through successive seed generations. However, the gene regulatory pathways underlying apomixis remain unknown. In particular, the potential function of microRNAs, which are known to play crucial roles in many aspects of plant growth and development, remains to be determined with regards to the switch from sexual to apomictic reproduction.ResultsUsing bioinformatics and microarray validation procedures, 51 miRNA families conserved among angiosperms were identified in Boechera. Microarray assay confirmed 15 of the miRNA families that were identified by bioinformatics techniques. 30 cDNA sequences representing 26 miRNAs could fold back into stable pre-miRNAs. 19 of these pre-miRNAs had miRNAs with Boechera-specific nucleotide substitutions (NSs). Analysis of the Gibbs free energy (ΔG) of these pre-miRNA stem-loops with NSs showed that the Boechera-specific miRNA NSs significantly (p ≤ 0.05) enhance the stability of stem-loops. Furthermore, six transcription factors, the Squamosa promoter binding protein like SPL6, SPL11 and SPL15, Myb domain protein 120 (MYB120), RELATED TO AP2.7 DNA binding (RAP2.7, TOE1 RAP2.7) and TCP family transcription factor 10 (TCP10) were found to be expressed in sexual or apomictic ovules. However, only SPL11 showed differential expression with significant (p ≤ 0.05) up-regulation at the megaspore mother cell (MMC) stage of ovule development in apomictic genotypes.ConclusionsThis study constitutes the first extensive insight into the conservation and expression of microRNAs in Boechera sexual and apomictic species. The miR156/157 target squamosa promoter binding protein-like 11 (SPL11) was found differentially expressed with significant (p ≤ 0.05) up-regulation at the MMC stage of ovule development in apomictic genotypes. The results also demonstrate that nucleotide changes in mature miRNAs significantly (p ≤ 0.05) enhance the thermodynamic stability of pre-miRNA stem-loops.

Project description:Apomictic plants (reproducing via asexual seeds), unlike sexual individuals, avoid meiosis and egg cell fertilization. Consequently, apomixis is very important for fixing maternal genotypes in the next plant generations. Despite the progress in the study of apomixis, molecular and genetic regulation of the latter remains poorly understood. So far APOLLO gene encoding aspartate glutamate aspartate aspartate histidine exonuclease is one of the very few described genes associated with apomixis in Boechera species. The centromere-specific histone H3 variant encoded by CENH3 gene is essential for cell division. Mutations in CENH3 disrupt chromosome segregation during mitosis and meiosis since the attachment of spindle microtubules to a mutated form of the CENH3 histone fails. This paper presents in silico characteristic of APOLLO and CENH3 genes, which may affect apomixis. Furthermore, we characterize the structure of CENH3 by bioinformatic tools, study expression levels of APOLLO and CENH3 transcripts by Real-Time Polymerase Chain Reaction RT-PCR in gynoecium/siliques of the natural diploid apomictic and sexual Boechera species at the stages of meiosis and before and after fertilization. While CENH3 was a single copy gene in all Boechera species, the APOLLO gene have several polymorphic alleles associated with sexual and apomictic reproduction in the Boechera genera. Expression of the APOLLO apo-allele during meiosis was upregulated in gynoecium of apomict B. divaricarpa downregulating after meiosis until the 4th day after pollination (DAP). On the 5th DAP, expression in apomictic siliques increased again. In sexual B. stricta gynoecium and siliques APOLLO apo-allele did not express. Expression of the APOLLO sex-allele during and after meiosis in gynoecium of sexual plants was several times higher than that in apomictic gynoecium. However, after pollination the sex-allele was downregulated in sexual siliques to the level of apomicts and increased sharply on the 5th DAP, while in apomictic siliques it almost did not express. At the meiotic stage, the expression level of CENH3 in the gynoecium of apomicts was two times lower than that of the sexual Boechera, decreasing in both species after meiosis and keep remaining very low in siliques of both species for several days after artificial pollination until the 4th DAP, when the expression level raised in sexual B. stricta siliques exceeding 5 times the level in apomictic B. divaricarpa siliques. We also discuss polymorphism and phylogeny of the APOLLO and CENH3 genes. The results obtained may indicate to a role of the CENH3 and APOLLO genes in the development of apomixis in species of the genus Boechera.

Dataset Information

Gene Function Rather than Reproductive Mode Drives the Evolution of RNA Helicases in Sexual and Apomictic Boechera.

Publications

Gene Function Rather than Reproductive Mode Drives the Evolution of RNA Helicases in Sexual and Apomictic Boechera.

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